1. Field of the Invention
The present invention is directed to a novel protecting group useful in the synthesis of oligonucleotide analogs.
2. Discussion of the Background
It is well-known that most of the bodily states in mammals, including most disease states, are effected by proteins. By acting directly or through their enzymatic functions, proteins contribute in major proportion to many diseases in animals and man.
Classical therapeutics has generally focused on interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to directly inhibit the production of proteins involved in disease by interacting with the messenger RNA (mRNA) molecules that direct their synthesis. These interactions have involved the hybridization of complementary, or antisense, oligonucleotides or oligonucleotide analogs to MRNA. Hybridization is the sequence-specific hydrogen bonding of an oligonucleotide or oligonucleotide analog to an MRNA sequence via Watson-Crick hydrogen bond formation. By interfering with the production of proteins involved in disease, it has been hoped to effect therapeutic results with maximum effect and minimal side effects.
The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides and analogs thereof is their stability to nucleases. It is unlikely that unmodified oligonucleotides containing phosphodiester linkages will be useful therapeutic agents because they are rapidly degraded by nucleases. Modified oligonucleotides which are nuclease resistant are therefore greatly desired.
Phosphorothioate and phosphorodithioate oligonucleotide analogs have one or both of the non-bridging oxygens of the natural phosphodiester linkage replaced with sulphur, respectively, are especially promising antisense therapeutics. These oligonucleotide analogs are highly resistant to nucleases, have the same charge as natural phosphodiester-containing oligonucleotides, and are taken up by cells in therapeutically effective amounts. Other promising oligonucleotide analogs are those containing a mixture of phosphodiester and phosphorothioate and/or phosphorodithioate linkages. For a description of sulfurized oligonucleotide analogs see, Baracchini et al, U.S. Pat. No. 5,510,239; Ecker, U.S. Pat. No. 5,512,438; Bennett et al, U.S. Pat. No. 5,514,788; and Ecker et al, U.S. Pat. No. 5,523,389.
Oligonucleotide analogs are conveniently synthesized with automated DNA synthesizers using phosphoramidite chemistry. This is a highly efficient approach to the synthesis of oligonucleotide analogs, with coupling yields typically greater than 99%.
A more recent method for the synthesis of oligonucleotide analogs is the "blockmer" approach. In blockmer synthesis, an oligonucleotide analog is made by the sequential coupling of short protected oligomers or blocks, e.g., a dinucleotide, on a solid support. This strategy offers several advantages over the conventional synthetic approach which involves the sequential coupling of monomeric nucleoside phosphoramidites. The number of synthesis cycles required to prepare an oligonucleotide analog is reduced, saving time and minimizing reagent consumption. Importantly, the blocks may be prepared on a large scale using inexpensive solution phase synthesis techniques. The blockmer approach is described in the following references: Ravikumar et al, WO 95/32980; WO 94/15947; Journal of organic Chemistry 1984, 49, 4905-4912; Helevetica Chimica Acta 1985, 68, 1907-1913; Chem. Pharm. Bull. 1987, 35, 833-836.
One of the most important elements of oligonucleotide analog synthesis is the selection of a protecting group for the internucleosidic phosphorous linkages during step-wise synthesis. Removal of this protecting group should be fast and proceed through a mechanism which avoids nucleophilic attack at the phosphorous atom, which would result in chain scission. In addition, protected phosphoramidite monomers should be easy to synthesize inexpensively on a large scale. Phosphoramidite monomers containing the most widely-used phosphorous protecting group, .beta.-cyanoethyl, are very expensive to produce on a large scale. In addition, the phosphitylating reagent used in preparing .beta.-cyanoethyl protected phosphoramidites is explosive, which further limits the use of this protecting group in large scale synthesis. It is therefore of prime importance to develop low-cost protected nucleoside analog phosphoramidites which couple efficiently (&gt;99%) during step-wise synthesis and may be deprotected quickly in high yield under standard conditions.